Medicament containing an effector of the glutathione metabolism together with α-lipoic acid for treating diabetes mellitus

ABSTRACT

The invention relates to a medicament containing an effector of the glutathione metabolism together with α-lipoic acid for treating diabetes mellitus. This medicament enables disturbances of the thiol-disulfide status or those that occur, for example, in diabetes mellitus to be treated simultaneously, separately or in a temporally graduated manner.

BACKGROUND OF THE INVENTION

This invention relates to the use of the combination of α-lipoic acidand effectors of the glutathione metabolism for the treatment ofdisruptions of the cellular thiol status and related illnesses.

The precise regulation of the thiol disulfide status represents one ofthe most important basic requirements of biological metabolisms. Thecentral regulating element within this system is the tripeptideglutathione, which reaches high intracellular concentrations (up to 10mM) in reduced form. In addition to glutathione, protein groups carryingthiol groups intracellularly and particularly in cell-membrane-bondedform are additional important components of the thiol disulfide statusof each cell.

The metabolism of the breakup of disulfide and the formation of thethiol groups, which is regulated by various classes of enzymes, isessential for any normal cell function as a result of the variety of thebiological functions of thiol in, among other things, cell growth anddifferentiation processes including programmed cell death as well ascell protection and decontamination mechanisms overall. Disruptions inthis system and changes in the concentration of thiol lead to seriousdisruptions in cell function, which remain locally limited only inisolated cases, and generally have an adverse effect on the entireorganism.

The involvement of a disrupted thiol disulfide status in acute andchronic diseases has been demonstrated in a number of experiments.

In neurodegenerative diseases such as Parkinson's Disease, for example,significant changes in the thiol metabolism have been demonstrated incertain nerve cells (Brain Res Rev 1997; 25:335-358). There are clearindications that as a result of this metabolic disruption, there is anincreased death of nerve cells in the functionally impaired areas of thebrain, the basal ganglia, which is primarily responsible for thesymptoms of the disease (Ann Neurol 1994; 36:348-355).

Reduced glutathione levels and a reduced intracellular glutathionecontent have also been found in the context of angiopathies and theirconsequences—arteriosclerosis and heart attack—in the endothelial cellsthat line the inner wall of the vessel. (Med Sci Res 1998:26:105-106).

Pulmonary diseases that involve a transformation of the pulmonary tissueare regularly connected with a glutathione deficit in the tissue. In apulmonary fibrosis of this type, the severity of the disease runsparallel to the thiol loss (Chin Chim Acta 1997; 265:113-119). Seriousinflammatory pulmonary diseases, investigated using the example of acutedifficulty of breathing in adults, were accompanied by a dysregulationof the thiol metabolism of the participating inflammatory cells(granulocytes) (Chest 1996; 109:163-166).

On the basis of the author's own tests, the immuno-competent defensivecells of the 15 bronchial system (alveolar macrophages) in smokers andin patients with chronic obstructive respiratory diseases exhibit aserious cellular thiol deficit. The severity of the disruption of thecellular thiol status is thereby directly correlated with restrictionsin lung function (Free Radic Biol Med 2000; 29:1160-1165).

Extensive research into the significance of the glutathione metabolismin viral infections has revealed both a poorer prognosis ofthiol-deficient cells based on a compromised cellular defense, as wellas an antiviral function of the glutathione that combats thereproduction of the virus (Proc Natl Acad Sci USA 1997; 94:1967-1972).

The author's own tests have shown that the cellular thiol disulfidemetabolism is seriously disrupted, particularly under the conditions ofseverely restricted kidney function and the resulting necessary renalreplacement therapy in the form of hemodialysis or peritoneal dialysis.This disruption results in, among other things, the extensive loss ofnormal cell functions, such as the phagocytosis capability of peritonealmacrophages or the activation of lymphocytes.

The human cellular immune system, which consists of the white bloodcells granulocytes, lymphocytes and monocytes, represents a system thatreacts particularly sensitively to a disruption in the thiol metabolism.

Minimal changes, particularly losses of cellular glutathione, cantrigger a cascade-like program for the self-destruction of the cells,the programmed cell death (apoptosis) (FASEB J 1998; 12:479-486). Inthis case, the thiol disulfide metabolism acts as a central switchingmechanism of an intact immune system, without which the organism wouldnot be viable.

In recent years, moreover, there have been increasing references to adamaged thiol metabolism in chronic kidney diseases (Ren Fail 1998; 20:117-124), anemias (Br J Haematol 1995; 91:811-819), premature newborns(Pediatr Pulmonol 1995; 20:160 166), hearing loss caused by noise (BrainRes 1998; 784:82-90), inflammatory bowel diseases (Gut 1998; 42:485-492)and particularly in diabetes mellitus (Metabolism: Clinical andExperimental 1998; 47(8):993-997).

Studies in the context of diabetes mellitus and associated metabolicdisruptions have demonstrated both a shift of the redox status at theexpense of reduced glutathione as well as an absolute reduction of thetotal glutathione pool (Free Radic Biol Med 1998; 24:699-704). In thesummary of the previous literature on the role of the disruption of thethiol disulfide status, it has been assumed that not only is there anaccompanying SH deficit as a consequence of the primary disease, whichis Type 1 or Type 2 diabetes, but that a dysregulation in the thiolmetabolism is at least one factor that triggers the disease. Aconvincing example has been provided, among other things, by theverification of the radical-induced destruction of the pancreaticB-cells (Diabet Med 2000; 17:171-180).

It is also known that the disease is accompanied by a number ofimmunological disruptions. The primary factor that has been identifiedis an imbalance of the immunoregulator cytokine, which is related tofunctional disruptions of the lymphocytes and macrophages, with aresulting significant increase in the sensitivity of the patients toinfections (Horm Metab Res 1998; 30:526-530).

The correction of a disrupted thiol metabolism thus acquires fundamentalimportance as a basic therapy in the treatment of a number of diseasesof different geneses, particularly, however, under the conditions ofdiabetes mellitus.

α-lipoic acid has been used relatively successfully in the form of aneuro-protective substance for the treatment of neuro-toxically causedparesthesia in the context of diabetic polyneuropathy (Diabetologica1995; 38: 1425-1433, Diabetes Res Clin Pract 1995; 29:19-26, Diab Care1999; 22:1296-1301, Drug Metab Rev 1997; 29:1025-1054, DE 43 43 592 C2).DE 44 47 599 C2 and EP 0 530 446 B1 also describe the use of α-lipoicacid in additional neuronal disruptions, including tinnitus andapoplectiform deafness.

In this case, the cytoprotective mechanism of action depends on theinfluence of the sugar-dependent protein modification (proteinglycolysis), on a reduction of neurotoxic ketogenesis, and finally onthe anti-oxidation function of the α-lipoic acid and its metabolites(Free Radic Biol Med 1995; 19:227-250).

This cell protection function has been investigated particularly fromthe point of view of the prevention of the oxidative transformation ofessentially unsaturated fatty acids. Such an inhibition of the lipidperoxidation, in addition to the use of the α-lipoic acid as aneuro-protection agent, represents the basis for an application as amedicament to protect the liver in the treatment of variousintoxications and liver diseases (Biochemistry 1998; 37:1357-1364).

It has also been shown that α-lipoic acid inhibits the reproduction ofthe HIV virus in different stages of its growth and may thus counteracta progression of the AIDS disease. The results of these laboratory testscan be applied to clinical studies only to a limited extent, however(FEBS-Lett 1996; 394:9-13). The same is true for the detection of ananti-inflammatory function of the substance for the insulin-producingislet cells of the pancreas (Agents Actions 1993; 38:60-65).

EP 0 812 590 A2 and EP 0 427 247 B1 disclose the use of α-lipoic acid asa cyto-protective, as an analgesic and as a medicament for the treatmentof inflammatory diseases.

The anti-oxidative properties of α-lipoic acid are based on its abilityto form chelates with metal ions and to eliminate radicals directly, aswell as on its function as a strong reducing agent. To perform thisreaction on an intracellular level, α-lipoic acid, even in reduced form,must be present in the form of dihydrolipoic acid. The transition from(disulfide) α-lipoic acid by means of reduction into the dithiol form ofdihydrolipoic acid for its part consumes reducing equivalents, wherebythis process is catalyzed by, among other things, the enzyme glutathionereductase (Gen Pharmacol 1997; 29:315-331). This process is apparentlythe cause of the unsatisfactory action of the substance in terms ofthiol restitution.

Ambroxol, i.e. trans-4-(2-amino-3,5-dibromobenzylamino)-cyclohexanehydrochloride, is administered in various forms as an expectorant in thetreatment of pulmonary and bronchial diseases (WO 96 33704, GB 2239242,WO 01 05378). Its use in the treatment of hyperuricemia is also knownfrom DE 35 30 761. The action of ambroxol as a mucolytic is based bothon a stimulation of surfactant production by the bronchial cells andparticularly on its ability to eliminate free radicals (Respir Med 1998;92:609-23). The anti-oxidative activity of the substance based on theseproperties was demonstrated primarily on pulmonary cells (Pharmacol1999; 59:135-141), but also in the context of inflammatory mechanisms(Inflamm Res 1999; 48:86-93). It is also known that through the use ofambroxol in high doses, enzymes that regulate the glutathione metabolismcan be influenced directly and peroxidative processes can be inhibitedin vitro (Arch Vet Pol 1992; 32:57-66).

SUMMARY OF THE INVENTION

The object of the present invention was therefore to make availablenovel medicaments containing thiol-reactive substances for the improvedstabilization of an impaired thiol disulfide status in the treatment ofdiabetes mellitus and for the restitution of the functional lossescaused by said disease.

The invention teaches that this object can be achieved by a medicamentcontaining ambroxol having the Formula I,

Its salts and/or prodrugs, together with α-lipoic acid, its salts and/orprodrugs as combination preparation for the simultaneous, separate ortemporally graduated treatment of a disturbance of the thiol-disulfidestatus in diabetes mellitus and clinical pictures in which a disruptionof the thiol-disulfide status of immune cells occurs. Additionaloptional feature are also described herein below, as are some ofpossible uses of the medicament.

According to the invention, effectors of the glutathione metabolism areused in combination with α-lipoic acid, its salts and/or its prodrugs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart illustrating the effect of the combination of α-lipoicacid and ambroxol on the intracellular thiol expression of lymphocytes.

FIG. 2 is a chart illustrating the effect of the combination of α-lipoicacid and ambroxol on the expression of lymphocytes of cell membranebound thiols.

FIG. 3 is a chart illustrating the effect of the combination of α-lipoicacid and ambroxol on the cellular activation status of T-celllymphocytes.

FIG. 4 is a chart illustrating the effect of the combination of α-lipoicacid and ambroxol in a time kinetic over 14 days.

FIG. 5 is a chart illustrating the effect of the combination of α-lipoicacid and ambroxol on the membrane-resistant thiol expression ofperitoneal macrophages.

FIG. 6 is a chart illustrating the effect of the combination of α-lipoicacid and ambroxol on the intracellular thiol expression of theperitoneal macrophages in time kinetics.

FIG. 7 is a chart illustrating the effect of the combination of α-lipoicacid and ambroxol in the membrane-bound thiol expression.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention teaches that as a result of the application of thecombination of α-lipoic acid and an effector of the glutathionemetabolism used according to the invention, there is a normalization ofthe primary reduced thiol status of immune cells. Not only does thethiol-stabilizing action of the combinations regularly exceed that ofthe use of α-lipoic acid or of the respective effectors individually,but super-additive effects have also been found. The restitution of thethiol status thereby comprises both intra-cellular thiols as well asmembrane-bonded SH groups and is thus an expression of a complexbiological regulation. This phenomenon is based on the fact that theeffectors of the glutathione metabolism on the one hand eliminateintermediary free radicals that occur, and on the other hand increasethe availability of reducing equivalents for the transformation of theα-lipoic acid from disulfide into reduced form, and thus enhance thesynthesis-inducing effect of the α-lipoic acid on the thiol disulfidestatus.

It also became clear that a thiol-increasing effect of the combinationof effectors of the glutathione metabolism and α-lipoic acid occurredonly in primarily thiol-deficient immune cells. Healthy immune cells,which do not have any alteration of the thiol disulfide status, did notreact with a further increase of the SH concentration.

The restitution of the thiol status of the immune cells was accompaniedby a normalization of functional parameters. This phenomenon related inparticular to the immuno-modulatory effects in the context of theactivation of T-lymphocytes.

It has also been demonstrated that the combinations used according tothe invention stabilize the thiol disulfide status of additional immunecells such as the peritoneal macrophages of patients who requireddialysis. The peritoneal macrophages from high-glucose peritonealdialysis fluids, prior to treatment with α-lipoic acid/ambroxol inaddition to a deficient thiol status, have an almost complete loss oftheir phagocytosis function, as well as a serious disruption ofdifferentiation and cytokine synthesis, which have been described ascauses of the high infection rates in these patients. These functionallosses were eliminated by the addition of the combinations according tothe invention.

This medicament is particularly appropriate for the treatment ofdiabetes mellitus as well as other clinical pictures in which there is adisruption of the thiol disulfide status of the immune cells. Thetreatment can therefore be administered simultaneously, in separateformulations, or in a temporally graduated manner.

The combination preparations used according to the invention can beadministered in the conventional pharmacological forms of administrationor in the form of an instillate, as well as prophylactically andtherapeutically. The effective dose must thereby be determined on acase-by-case basis. The dose for applications in human patients ispreferably between 30 and 1200 mg/d, with particular preference given todoses between 200 and 600 mg/d.

In one variant, ambroxol having the general formula I

its salt and/or its prodrug are used as the effector of the glutathionemetabolism. The dose of ambroxol for applications in human medicine istherefore preferably between 7.5 and 90 mg/d, with particular preferencegiven to doses between 60 and 75 mg/d.

The medicament can thereby he administered orally and/or parenterally.

The medicament can also contain the conventional additives. Suchadditives include, for example, aqueous solvents, stabilizers,suspension agents, dispersion agents and wetting agents.

The medicament can be manufactured in any desired formulation. By way ofexample, acceptable formulations include solutions, granulates, powder,emulsions, tablets and/or coated tablets.

According to the invention, an effector of the glutathione metabolismtogether with α-lipoic acid, its salt and/or its prodrugs, are used forthe manufacture of a medicament for the treatment of a disruption of thethiol disulfide status of immune cells in the treatment of diabetesmellitus.

Likewise, an effector of the glutathione metabolism together withα-lipoic acid, its salt and/or its prodrugs can be used for themanufacture of a medicament for a treatment to modulate immunity,increase defenses or inhibit inflammation.

The components of the combination preparation can therefore be in asingle formulation or in separate formulations.

The use of the combination of α-lipoic acid and effects of theglutathione metabolism according to the invention is described ingreater detail below with reference to examples and the accompanyingfigures.

Example 1 Influence on the Cellular Thiol Status of Human PeripheralImmune Cells

Peripheral immune cells from healthy donors (n=9) were isolated from theperipheral blood. The major fraction of the resulting total populationof mononuclear cells regularly represents lymphocytes with a relativepercentage of approximately 90%, depending on the donor. 10% of themononuclear cells are represented by monocytes.

The mononuclear cells obtained were absorbed in special cell culturemedia and 25 incubated in a gasifying incubation cabinet at 37° C., arelative humidity of 98% and 5% relative air-CO₂ content. The metabolismof the primarily inactive immune cells was activated by means ofmitogenic stimulation (0.5 μg/ml phytohemagglutinin). To test theinfluence of the combinations used according to the invention on thethiol status of thiol-deficient immune cells, these cells wereartificially thiol-depleted. This process was carried out using provenmethods by cultivation in thiol-deficient media (RPMI 1603). Comparativecultures using complete media (RPMI) 1640) were used to define the bestpossible normal value under the culture conditions.

The determination of the intercellular thiol content on the single-celllevel was made using 5-chloromethyliluorescein diacetate (CMFDA) in flowcytofluorimetry.

Primarily non-fluorogenic CMIFDA is thereby passively absorbed by thecell. By means of the chlormethyl group, there is a bonding tocytoplasmic thiol groups. After the breakdown of the acetate groups bynon-specific cellular esterases, this complex, which is now cellmembrane bound, becomes fluorogenic at an excitation wavelengthλ_(ex)=490 nm with an emission wavelength λ_(em)=520 nm. The medianfluorescence intensity of the specimen (10,000 cells) is directlyproportional to the concentration of the intracellular thiol groups.

The expression of membrane-bound thiol groups was also determined usingflow cytofluorimetry. In this case, chloromethyltetramethyl rhodamine(CMTMR) was used as thiol conjugate under the conditions of a blockedmembrane potential and an inhibited diffusion capacity of the cells. Thefluorescence intensity of the fluorochrome molecules bound to the cellmembrane is in turn proportional to the number of thiol groups on thecell surface.

FIG. 1 illustrates the effect of the combination of α-lipoic acid andambroxol on the intracellular thiol expression of lymphocytes. The dataare presented as the ratio of the cellular fluorescence intensity to therespective calibration particles (beads) that were analyzedsimultaneously. The actives concentration of the respective combinationis identical to the concentrations of the individual components.

Peripheral immune cells were cultivated over a period of 4 days understandard (Control 1640) or thiol-deficient conditions (1603) for theinduction of a 10-20% thiol reduction. As illustrated in 1, the additionof ambroxol in combination with α-lipoic acid beginning after 48 hoursof treatment resulted in a total compensation of the intracellular thioldeficit. It was not possible to achieve a complete compensation of thethiol deficit using either α-lipoic acid alone or with the individualapplication of the effector.

The results obtained with this experimental investigation of theinfluence of the combinations according to the invention on theexpression of cell membrane bound thiols are presented in FIG. 2 for thecombinations of α-lipoic acid and ambroxol. In the treatment using thecombination of α-lipoic acid and ambroxol, there was once again,beginning after 48 hours, a significant improvement in themembrane-bound thiol expression. In this case, it was particularlynoteworthy that the addition of the individual substances did not at anytime have a significant effect

Example 2 Influence on the Cellular Activation Status of HumanPeripheral T-Lymphocytes

In the cultivation experiment described with reference to Example 1,human T-lymphocytes were stimulated with 1.0 μg/ml phytohemagglutinin.In a culture time of 72 hours, specific markers of the cellularactivation were quantitatively detected using cytofluorimetery by meansof monoclonal antibodies. The influence of the combinations usedaccording to the invention on the activation markers CD69 (earlyactivation antigen), CD25 (intermediate activation antigen) and CD71(late activation antigen) of T-lymphocytes was measured. FIG. 3 showsthe effect of the combination of α-lipoic acid and ambroxol on theactivation index of T-lymphocytes. Compared with normal T-lymphocytes(activation index=1.0), in thiol-deficient cells there is a clearreduction of the activation, which demonstrates the disruption ofcellular function. After the addition of α-lipoic acid, the well-knowneffect of a slight improvement of the cellular activation occurs, whichhowever in no case caused any significant difference from the normalcontrol group. Ambroxol does not have any influence on one of the threeactivation markers. On the other hand, with the combined use of bothα-lipoic acid and ambroxol, there was a detectable increase in theT-cell activation index in the normal range. This effect was observedwith early, intermediate and hate activation markers. It can thus beconcluded that the normalization of the cellular thiol status achievedby the combined use of α-lipoic acid and the respective effector of theglutathione metabolism is accompanied by a restitution of cell function.

Example 3 Influence on the Cellular Thiol Status of PeritonealMacrophages in the Context of Renal Replacement Therapy

Peritoneal macrophages were isolated from the effluate of peritonealdialysis of patients with a high degree of renal insufficiency, absorbedin a cell culture medium and incubated in a gasifying incubation cabinetat 37° C., a relative humidity of 98% and 7.5% relative air-CO₂ content.To investigate the influence of the combinations used according to theinvention on the thiol status of the peritoneal macrophages, individualfractions were treated with α-lipoic acid, the effectors of theglutathione metabolism ambroxol and with the combination of α-lipoicacid/ambroxol, while an additional fraction was maintained as anuntreated control.

The cellular thiol status was determined using the measurement methodsdescribed in Example 1.

FIG. 4 illustrates the effect of the combination of α-lipoic acid andambroxolin a time kinetic over 14 days (n=12).

When the individual substances were added, there was again an increaseof the cellular thiol expression only when α-lipoic acid was used, whileambroxol had no effect. On the other hand, with the combination ofα-lipoic acid and ambroxol, beginning after 72 hours, there was asignificant increase of cellular thiol expression, which achieved asuper-additive effect after 4 days of treatment and a maximum after 8days, which exceeded the initial or control data by a factor of three(FIG. 4).

FIG. 5 shows the effect of the combination of α-lipoic acid andenalapril (FIG. 5 b) on the membrane-resistant thiol expression ofperitoneal macrophages in the experimental system described above. Themembrane expression of thiols was determined on the basis of the medianfluorescence intensity (mfi) of the specimen (3000 cells/measurement)after coupling to a chlormethyl-fluorochrome derivative.

In comparison with the results of the intracellular thiol expression, inthis case there is a very clear effect of the addition of α-lipoic acidby itself, although that effect disappeared after 4 days of treatment.In contrast, the combined application of α-lipoic acid and ambroxolresulted in both a primarily more significant super-additive increase ofthe membrane-bound thiol expression, and one that was more stable overthe observation period.

Example 4 Influence on the Phagocytosis Capacity of PeritonealMacrophages

Phagocytosis capacity was selected as a measurement to make it possibleto characterize the peritoneal macrophages with regard to their originalfunctions.

Peritoneal macrophages were isolated using a method analogous to themethod described in Example 3 and cultivated ex vivo.

The phagocytosis capacity was determined by a cytofluorimetric test onthe single-cell level. The macrophages were thereby co-cultivated withopsonized and fluorochrome-marked bacteria. The number of bacteriaabsorbed during a defined period was determined quantitatively by meansof the fluorescence intensity in the macrophages and was used as ameasurement for their phagocytosis capacity.

The influence of the combinations used according to the invention on thephagocytosis capacity of the peritoneal macrophages after a treatmenttime of 6 days is presented in the following table.

Phagocytosis rate (mfi / 10,000 cells) Control 371 ± 39 α-lipoic acid[50 μM] 687 ± 59 Ambroxol [10 μM] 501 ± 52 α-lipoic acid + ambroxol1,398 ± 286  (p < 0.05)

After incubation with α-lipoic acid, ambroxol, the phagocytosis rate washigher than that of the untreated control by a factor of 1.85 (α-lipoicacid), 1.35 (ambroxol). On the other hand, when the combination ofα-lipoic acid and ambroxol was used, there was an increase in thephagocytosis rate by a factor of 3.7.

Moreover, a direct correlation was established between the phagocytosisrate and the intracellular thiol content of the peritoneal macrophagesfor the combination of α-lipoic acid and ambroxol (r=0.79; p<0.01).

Example 5 Influence on the Degree of Differentiation and Activation andthe Cytokine Synthesis of Peritoneal Macrophages

Peritoneal macrophages were isolated from patients during renalreplacement therapy using the method described in Example 3 andcultivated in the presence of the combinations of α-lipoic acid andeffector of the glutathione metabolism according to the invention. After6 days of incubation, the degree of differentiation of the peritonealmacrophages was determined by means of the expression of the cellsurface antigens CD15 and CD11c, and the degree of cellular activationwas determined using cytofluorimetry by means of the co-expression ofthe activation antigens CD69 to CD15-positive cells and CD71 toCD11c-positive cells.

The results are presented in the following table:

CD15 CD11c CD15/69 CD11c/71 Control 1.0 1.0 1.0 1.0 α-lipoic acid 1.18 ±0.16 1.21 ± 0.11 1.09 ± 0.08 1.08 ± 0.09 [50 μM] Ambroxol 0.98 ± 0.131.01 ± 0.09 0.09 ± 0.11 0.96 ± 0.1  [10 μM] α-lipoic acid + 1.29 ± 0.211.65 ± 0.21 1.49 ± 0.13 1.83 ± 0.14 ambroxol

It was shown that the expression of the maturation markers CD15 andCD11c increased markedly with the use of the combination of α-lipoicacid and ambroxol. There was also a significant increase in theactivation antigens CD69 and CD71 respectively in the respective cellpopulations. The application of the single substances had no effect oronly a marginal effect on the degree of differentiation and activationof peritoneal macrophages.

Simultaneously, in this experimental approach the cell culture residuewas collected and the cytokines Interleukin-6 (IL-6) and Interleukin-1receptor antagonist (IL-1ra) synthesized and secreted by the peritonealmacrophages contained in the residue were determined. The analysis wasperformed using the enzyme immunoassay technique with standardizedmeasurement systems.

There was a significant reduction of the IL-6 synthesis in the presenceof the combination of α-lipoic acid and ambroxol. This effect in turnwent significantly beyond the sum of the reduction achieved by theindividual substances. The synthesis of IL-1ra under these conditionswas significantly induced. In this case, too, there was a super-additiveeffect of the combination of α-lipoic acid and ambroxol.

Example 6 Influence on the Stability of Thiol Restitution in PeritonealMacrophages in the Dialysis Model

The combinations of thiol-restored peritoneal macrophages used accordingto the invention were extracted from this test system after 6 days andcultivated over a period of 14 days in a dialysis model. For thispurpose the peritoneal macrophages were adapted to matrices that werecoated with collagen IV and placed in contact with conventionalhigh-glucose dialysis solution 3 times a day for 60 minutes each time.In this case, the model was used for the induction of a combinedhyperglycemic/osmotic stress. FIG. 6 shows the effect of the combinationof α-lipoic acid and ambroxol on the intracellular thiol expression ofthe peritoneal macrophages in time kinetics. The membrane expression ofthiol was determined on the basis of the median fluorescence intensity(mfi) of the specimen (3000 cells/measurement) after coupling to achlormethyl-fluorochrome derivative. While with the primarilythiol-restored controls that had not been treated in this dialysismodel, there was a practically linear reduction of the intracellularthiol concentration within the first 4 days, the combined addition ofα-lipoic acid and ambroxol resulted in a constant intracellular thiolstatus on the level of the primary restitution. Here, too, there is amono-effect of α-lipoic acid, although it lasts only briefly and afterapproximately 4 days in the dialysis model is only approximately 50% aseffective as the combinations.

A similar result is apparent in the curves of the membrane-bound thiolexpression illustrated in FIG. 7. Here again, the quantities obtained bythe primary thiol restitution are kept constant by the use of thecombination of α-lipoic acid and ambroxol, while with the addition ofthe individual substances, only intermediary (α-lipoic acid) or marginaleffects (ambroxol) were observed.

The effects of α-lipoic acid and effector of the cellular glutathionemetabolism on the cytokine synthesis of peritoneal macrophages after atreatment of 6 days (n=10) are presented in the following table.

IL-6 IL-1ra [ng/10⁶ cells] [ng/10⁶ cells] Control 53.1 ± 8.9 115.2 ±23.4 α-lipoic acid [50 μM] 46.9 ± 6.7 119.8 ± 19.5 Ambroxol [10 μM] 51.8± 8.1 118.6 ± 21.3 α-lipoic acid + ambroxol 31.5 ± 9.2 126.8 ± 15.3 (p <0.05) (p < 0.05)

Overall, these tests make it plain that the application of thecombination of α-lipoic acid and the effectors of the glutathionemetabolism ambroxol stabilizes a primarily massively damaged thiolstatus in different cell systems. As a result of this normalization,there is also a re-establishment of central cellular immuno-regulatoryfunctions, which is not achieved without such a treatment.

1. A method for the treatment of a known symptom of diabetes mellitus,namely a disturbance of the membrane-bound thiol disulfide status inperipheral immune cells, comprising the step of administering a compoundselected from the group consisting of ambroxol having the Formula I,

its salts and mixtures thereof in a dosage range of 7.5 mg/d and 90mg/d, between 60 mg/d and 75 mg/d, and a compound selected from thegroup consisting of α-lipoic acid, its salts and mixtures thereof in adosage range of 30 mg/d and 1200 mg/d, or between 200 mg/d and 600 mg/d.2. A method for the treatment of a known symptom of diabetes mellitus,namely a disturbance of the intracellular thiol disulfide status inperipheral immune cells, comprising the step of administering a compoundselected from the group consisting of ambroxol having the Formula I,

its salts and mixtures thereof in a dosage range of 7.5 mg/d and 90mg/d, between 60 mg/d and 75 mg/d, and a compound selected from thegroup consisting of α-lipoic acid, its salts and mixtures thereof in adosage range of 30 mg/d and 1200 mg/d, or between 200 mg/d and 600 mg/d.3. A method for treatment of a disturbance of the thiol disulfide statusaccording to any of claims 1-2, where the cells described are peripheralimmune cells of patients with diabetes mellitus.
 4. A method for theactivation of thiol-deficient T-cells, including the step ofadministering a compound selected from the group consisting of ambroxolhaving the Formula I,

its salts and mixtures thereof in a dosage range of 7.5 mg/d and 90mg/d, between 60 mg/d and 75 mg/d, and a compound selected from thegroup consisting of α-lipoic acid, its salts and mixtures thereof in adosage range of 30 mg/d and 1200 mg/d, or between 200 mg/d and 600 mg/d.5. Method of treatment as recited in any one of claims 1, 2, 4, whereinthe medicament is administered orally or parenterally.
 6. Method oftreatment as recited in any one of claims 1, 2, 4, wherein themedicament contains additional additives selected from the groupconsisting of aqueous solvents, stabilizers, suspension, dispersion andwetting agents.
 7. Method of treatment as recited in any one of claims1, 2, 4, wherein the medicament is in the form of a solution, agranulate, a powder, an emulsion, a tablet and/or coated tablet.